Many of the interactions between cells in multicellular organisms involve the secretory and endocytic apparatus. This apparatus consists of the plasma membrane and intracellular membrane-bound organelles. It is involved in neural and endocrine signaling and in cell adhesion. The apparatus is also important in cellular nutrition and in determ1ning the composition of the extracellular space. The two projects in this proposal will explore different aspects of the complex processes of secretion and endocytosis. The first project seeks to understand the intracellular movement of receptors during receptor-mediated endocytosis. To accomplish this, we are developing novel biochemical assays for intracellular transport. These techniques will allow us to follow receptors as they enter the cell in ligand-receptor complexes, deliver their ligands to lysosomes and return to the cell surface. Studies will concentrate on two molecules: transferrin receptor, the receptor involved in iron uptake by animal cells, and mannose-6-phosphate receptor, the receptor involved in packaging lysosomal enzymes into lysosomes. The transport of receptors into the Golgi, the endoplasmic reticulum (ER) and lysosomes will be studied. Results should reveal the importance of this transport in receptor function. We will also be able to map out routes of membrane traffic between the cell surface and intracellular organelles. The second project will study the export of sugar residues from the cytoplasm during the synthesis of asparagine-linked oligosaccharides in the ER. We have proposed that sugars cross the ER membrane during this process by the reorientation of oligosaccharide-lipid precursors. We plan to demonstrate the transmembrane movement of oligosaccharide-lipids directly and identify membrane proteins that catalyze this reorientation. These studies will show how this translocation causes the coupling of glycoprotein synthesis and secretion in the ER. Similar transmembrane movement of large intermediates occurs during the snythesis of all secreted macromolecules. Our experiments on asparagine-linked oligosaccharide assembly should make this the first system for which molecular and mechanistic details of this movement are understood.